Stabilization of Lecitase Ultra^® by Immobilization and Fixation of Bimolecular Aggregates. Release of Omega-3 Fatty Acids by Enzymatic Hydrolysis of Krill Oil

Round 1

Reviewer 1 Report

The authors tried to improve the manuscript. However, I still have some concerns about the manuscript publication. The first issue is the manuscript style. Usually, the “introduction” section is aimed at introducing the reader to the scientific problem. The “Introduction” of the present manuscript is in large part a work schedule, some paragraphs are to be moved in the Materials and methods section.

The main concern is about the experimental strength of the reported hypothesis.  The authors' interpretation of the experimental data is questionable. A few representative examples:

Page 5:

The authors observe that the presence of a surfactant (CTAB) slows down the immobilization rate of the enzyme on PEI-agarose. The authors argue that this is due to the effect of CTAB on the enzyme aggregation, since  “If immobilization occurs through only one lecitase molecule, the immobilization rate should be similar in the presence or absence of CTAB”. How can the authors exclude other effects? CTAB is a surfactant that can interact with all the reagents, particularly with the PEI-agarose support.

Page 6 :

“This multipoint covalent attachment can promote enzyme distortions, but can highly stabilize the 3D structure. This may be the cause of the highly increased stability as well as the relevant decrease in enzyme activity commented above. “

Since no substantial proof is reported, this sentence is rather questionable. If a covalent multipoint attachment occurs, the 3D structure should be denatured, not stabilized.

Finally, many typos and the Tables are almost unreadable due to the wrong page layout and the lack of fundamental scientific details. In Table 1 the cells reporting the half-life time lack of the unit of measure.

Author Response

• A new process for immobilization-stabilization of Lecitase is here proposed. The main points of this new strategy should be remarked out in Introduction. As suggested by Reviewer, Introduction has been shortened.
• The surfactant CTAB was used with a very small concentration (2.7 mM). In addition, it has a positive charge and the support has also positive charges. In this way, interaction of CTAB with the support should be minimal. Very likely, the main effect of CTAB is the dissociation of the bimolecular aggregate. “Very likely” is now included to remark the different behavior between Lecitase with and without CTAB.
• Multipoint covalent immobilization promotes distortions of the enzyme structure but it also greatly stabilizes the distorted structure. A number of residues are attached to the support with fixed relative positions of the distorted enzyme and this immobilized distorted enzyme should be highly stabilized
• All other mistakes have been corrected
• English has been edited by MDPI

Reviewer 2 Report

In this article, the authors reported “Immobilization and fixation of bi-molecular aggregates of open structures of Lecitase to different immobilization protocols including Covalent immobilization CNBr-agarose in the presence and absence of surfactant (CTAB) as well as Ionic adsorption on PEI-agarose in the absence of CTAB and Interfacial adsorption on 
octyl-agarose, their stabilization and their application to enzymatic hydrolysis of krill oil.

Recovery activity of the immobilized enzyme also demonstrated

The article is well written and structured

Please adjust the tables and figures to Catalysts journal format

I believe this article is suitable for publication in Catalysts

Author Response

Tables and Figures were adjusted to the journal format

Reviewer 3 Report

The paper reports different protocols to immobilize Lecitase Ultra a chimeric enzyme used for degumming krill oil.  The topic is of interest for Catalysts and the design of experiments  appropriate.

However the paper should describe better how to discriminate between the closed and open form. Is any spectroscopic method (es CD) available? In addiction why it is not possible to link directly the closed form in diluted solutions instead of  linking the open form in presence of tensioactives and then removing them?

The numeration of References is completely wrong.

Minor typographical errors are present in the text.

Author Response

The fact that the bimolecular aggregate contains two open structures is clearly supported. Aggregates are dissociated by CTAB and the main hydrophobic regions of Lecitase are the open active centers. In previous papers (commented in this one) lipases are adsorbed on other immobilized lipase. The main logical mechanism is the interaction between open hydrophobic structures

Very dilute enzyme solutions are mainly dissociated but they would promote lowly loaded enzyme derivatives. The use of more concentrated enzyme solutions in the presence of CTAB promotes a similar effect and allows the preparation of highly loaded derivatives of the isolated Lecitase molecule.

Numeration of References has been improved

Typographical errors have been corrected

Reviewer 4 Report

In the paper presented by Andres-Sanz et al., the authors studied immobilization and fixation of bimolecular aggregates to the support surface. The most stable derivative was obtained by covalent immobilization of the bimolecular aggregate. They found that immobilized derivatives of Lecitase aggregates were much more stable than diluted solutions of soluble enzyme. After the immobilization, the authors tested enzyme in the reaction of hydrolysis of omega-3 fatty acids.  

Although the paper has potential and it is interesting, due to bad organization of the paper and low quality of presentation that was lost. In order to improve quality of the paper, fallowing issues must be addressed:

Major:

1. Title sounds more like 2 highlights then a title. New, comprehensive title that describes the paper should be added.
2. Abstract also needs improvement. It should be in the form: background, main objective, main results.
3. Negative aspects of immobilization should also be discussed in introduction.
4. page 1, second row from the bottom.” Lecitase is very useful for oil degumming [3,4], resolution of racemic mixtures [5], asymmetric hydrolysis [6], synthesis of phospholipids, etc.”: reference for synthesis of phospholipids is missing
5. Using words like “We will study” “our hypothesis” etc. is usually avoided in scientific papers. Should be “The aim of this study …” and “The main hypothesis are…”. Fourth paragraph on 2 page is a good example how not to do it.
6. Overall, introduction is written poorly. There is hardly background of the research, paragraph 4 on the second page is strange – usually when the aim of the work is written it is not in the form: “If something will be obtained then we will try to do this and this…” Hypotheses are welcome in the introduction but this form is strange. It is too long with too much irrelevant information. I suggest rewriting the whole introduction first focusing on the application of the enzyme, then the advantages and disadvantages of immobilization. Literature background on known methods in correlation with studied enzyme. Limitations of those studies and solution that authors present in this paper. Hypothesis can be placed in this section but in short bullets. More about them can be discussed in the results. What were the expectations and what were the outcomes.
7. Why are all figures in separate chapter? It is really hard to fallow the paper in this form. They should be rearranged and appear in the paper near the text where they are mentioned for the first time
8. Why the hydrolysis was performed at room temperature and at pH 7 since most of lipases have higher pH and temperature optimum? Enzyme characteristics should be mentioned in order to avoid such questions.
9. What is room temperature? I guess 25°C but authors should be more precise.
10. Language needs proofing. There are many mistakes.
11. Authors should avoid subjective statements like very interesting
12. Deactivation constant can be easily calculated from obtained data (Figure 7).
13. Figure 8 is not mentioned in the manuscript
14. Figure 2 is repeating in Table 4. One should be removed
15. 5.1. a reference of the method should be provided.
16. overall, references are to old. Provide examples from newer research papers  

Minor:

1. Why use capital letters for Chemical Industry or Enzymatic Engineering?
2. Thermomyces lanuginosus should be written in italic
3. page 1, second row from the bottom. [ should be removed. ([The preparation of heterogeneous and robust biocatalysts of Lecitase has a lot of potential.)
4. page 1. First row. “to Lecitase immobilization [4,5.8-11]:” Dot should be removed from the reference and replaced with comma. Also : should be removed from the end of the sentence and replaced with dot
5. Format of Table 1 Is strange
6. Table 3 is broken
7. 3. and 3.4 section. Please avoid using we
8. section 3.4. extinction coefficient should be in italic, and -1 should be in superscript. Word Enzyme should be written with small letter.
9. 5.1. H2O, 2 should be in subscript
10. If the name of the enzyme is Lecitase-Ultra® that name should be used in complete manuscript.
11. Buffer…. it is written in 2 forms: 25 mM phosphate buffer pH 7 and phosphate buffer (25 mM, pH=7). Make it uniform.
12. Section 3.9. – End of this paragraph.. Conclusion is in the sentence… should be removed

Author Response

Reviewer 4

In the paper presented by Andres-Sanz et al., the authors studied immobilization and fixation of bimolecular aggregates to the support surface. The most stable derivative was obtained by covalent immobilization of the bimolecular aggregate. They found that immobilized derivatives of Lecitase aggregates were much more stable than diluted solutions of soluble enzyme. After the immobilization, the authors tested enzyme in the reaction of hydrolysis of omega-3 fatty acids.  

Although the paper has potential and it is interesting, due to bad organization of the paper and low quality of presentation that was lost. In order to improve quality of the paper, fallowing issues must be addressed:

Major:

1. Title sounds more like 2 highlights then a title. New, comprehensive title that describes the paper should be added.

The title has been simplified. The two topics (Biocatalyst engineering and Biotransformation) are maintained according to the topic od the Special Issue

1. Abstract also needs improvement. It should be in the form: background, main objective, main results.

Abstract have been improved

1. Negative aspects of immobilization should also be discussed in introduction.
2. page 1, second row from the bottom.” Lecitase is very useful for oil degumming [3,4], resolution of racemic mixtures [5], asymmetric hydrolysis [6], synthesis of phospholipids, etc.”: reference for synthesis of phospholipids is missing

synthesis of phospholipids has been suppressed

1. Using words like “We will study” “our hypothesis” etc. is usually avoided in scientific papers. Should be “The aim of this study …” and “The main hypothesis are…”. Fourth paragraph on 2 page is a good example how not to do it.

Those mistakes have been corrected

1. Overall, introduction is written poorly. There is hardly background of the research, paragraph 4 on the second page is strange – usually when the aim of the work is written it is not in the form: “If something will be obtained then we will try to do this and this…” Hypotheses are welcome in the introduction but this form is strange. It is too long with too much irrelevant information. I suggest rewriting the whole introduction first focusing on the application of the enzyme, then the advantages and disadvantages of immobilization. Literature background on known methods in correlation with studied enzyme. Limitations of those studies and solution that authors present in this paper. Hypothesis can be placed in this section but in short bullets. More about them can be discussed in the results. What were the expectations and what were the outcomes.

Hypothesis has been simplified

1. Why are all figures in separate chapter? It is really hard to fallow the paper in this form. They should be rearranged and appear in the paper near the text where they are mentioned for the first time

Figures have been integrated in the text

1. Why the hydrolysis was performed at room temperature and at pH 7 since most of lipases have higher pH and temperature optimum? Enzyme characteristics should be mentioned in order to avoid such questions.

Omega-3 fatty acids are very instable. Very mild reaction conditions are required

1. What is room temperature? I guess 25°C but authors should be more precise.

Room temperature has been replaced by 25°C

1. Language needs proofing. There are many mistakes.

Language has been edited by MDPI

1. Authors should avoid subjective statements like very interesting

Subjective statements have been replaced

1. Deactivation constant can be easily calculated from obtained data (Figure 7).
2. Figure 8 is not mentioned in the manuscript

1. Figure 2 is repeating in Table 4. One should be removed
2. 5.1. a reference of the method should be provided.

A reference has been provide

1. overall, references are to old. Provide examples from newer research papers  

There are not recent papers related to lipase aggregation

Minor:

1. Why use capital letters for Chemical Industry or Enzymatic Engineering?

Capital letters have been removed

1. Thermomyces lanuginosus should be written in italic

OK

1. page 1, second row from the bottom. [ should be removed. ([The preparation of heterogeneous and robust biocatalysts of Lecitase has a lot of potential.)

It has been removed

1. page 1. First row. “to Lecitase immobilization [4,5.8-11]:” Dot should be removed from the reference and replaced with comma. Also : should be removed from the end of the sentence and replaced with dot

OK

1. Format of Table 1 Is strange

Format has been improved

1. Table 3 is broken
2. 3. and 3.4 section. Please avoid using we

Ok

1. section 3.4. extinction coefficient should be in italic, and -1 should be in superscript. Word Enzyme should be written with small letter.

OK

1. 5.1. H2O, 2 should be in subscript

OK

1. If the name of the enzyme is Lecitase-Ultra® that name should be used in complete manuscript.

It is a bit complex. The name is explained the first time

1. Buffer…. it is written in 2 forms: 25 mM phosphate buffer pH 7 and phosphate buffer (25 mM, pH=7). Make it uniform.

OK

1. Section 3.9. – End of this paragraph.. Conclusion is in the sentence… should be removed

Ok

Round 2

Reviewer 3 Report

The paper can be accepted for publication

Author Response

OK, Thank you very much

Reviewer 4 Report

After reading authors replay to reviewers comment conclusion is that the paper has improved but there are still some questions that need to be addresses.

1. row 30. Thermomyces lanuginosus should be in italic
2. Paragraphs 36-45 and 46-56 are similar and should be combined in one paragraph and placed at the end of Introduction
3. I still don’t see the point of section hypothesis. This should be merged with previous text in the introduction
4. hypothesis 1.3. and 1.4 It is strange to mention experimental designee in the introduction. Advice is to avoid this
5. figure 6 . y axis should be relative activity.
6. Figure 6 and 7 to make figures uniform please use same style when drawing lines
7. 4. Release of omega 3 fatty acids should be omega- 3

Author Response

there are still some questions that need to be addresses.

1. row 30. Thermomyces lanuginosus should be in italic

This mistake has been corrected

1. Paragraphs 36-45 and 46-56 are similar and should be combined in one paragraph and placed at the end of Introduction

They have been combined and placed at the end of Introduction

1. I still don’t see the point of section hypothesis. This should be merged with previous text in the introduction

The strategies for immobilization and fixation of bimolecular aggregates of Lecitase are novel and interesting and they should be explained in Introduction. Otherwise, experimental protocols and results cannot be understood.

1. hypothesis 1.3. and 1.4 It is strange to mention experimental designee in the introduction. Advice is to avoid this

The immobilization of concentrated Lecitase as a monomolecular structure has to be discussed. This is not found in other papers and it is novel and interesting.

The possible  release of omega-3 fatty acids by a phospholipase A1 has to be discussed in Introduction. Otherwise the experimental protocol would be erroneous. In the absence of migration issues the release of fatty acids placed in the sn2 position with a phospholipase A1 would be difficult to understand.

All these hypotheses are also useful for most of lipases

1. figure 6 . y axis should be relative activity.

This has been corrected

1. Figure 6 and 7 to make figures uniform please use same style when drawing lines

Figures have been improved and uniformed

1. 4. Release of omega 3 fatty acids should be omega- 3

1. This has been corrected

Round 3

Reviewer 4 Report

The authors have adequately revised the manuscript according to reviewer´s comments and given appropriate explanation for reviewer´s questions. All new contributions improve the manuscript quality.